Diabetologia (1982) 23: 65-68 Diabetologia �9 Springer-Verlag 1982

Short Communications

Alterations of Exocrine Pancreatic Enzymes in Virus-Induced Diabetic Cattle as Revealed by Immunohistochemistry

M. Bendayan* , S. I to** and I. M a n o c c h i o 1

Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland and llstituto di Patalogia Generale e Anatomia Patologica Veterinaria, Universitfi degli Studi, Perugia, Italy

Summary. The pancreatic tissue of normal and virus-induced diabetic cattle was investigated by the indirect immunofluores- cence technique. Seven secretory proteins (chymotrypsinogen A, trypsinogen, carboxypeptidase A, RNase, DNase, a-amy- lase and lipase) were localized in normal bovine pancreatic act- nat cells but in diabetic animals amylase, lipase and carboxy- peptidase were either not detectable or markedly diminished.

Decrease in amylase content has been reported previously in other diabetic animals. The diminution of the three pancreatic enzymes may be related to the destruction of pancreatic endo- crine tissue that occurs in these diabetic animals.

Key words: Bovine exocrine pancreas, pancreatic enzymes, dia- betes, immunofluorescence, cattle.

In previous studies [7, 8], we were able to reveal different digestive enzymes in the pancrea t ic ac inar cells o f the no rma l rat. By using immunof luo re scence , al terations o f the endocr ine pancreas in diabetes have been d o c u m e n t - ed [1, 15, 19] but, in contrast , little is k n o w n abou t the changes occur r ing in the exocr ine pancreas . I n diabet ic pat ients the exocr ine secret ion is impa i red [9, 20], and in a l loxan-diabet ic rats, Ben Abdeljli l et al. [4] repor ted that a l loxan reduces the amylase conten t in the pancreas bu t increases the level o f chymot ryps inogen . In the present s tudy, no rma l and v i rus- induced diabet ic bovine exo- crine pancreases were invest igated using specific anti- e n z y m e antisera and an i m m u n o f l u o r e s c e n c e technique descr ibed previous ly [7].

Materials and Methods

Preparation of the Antisera

Seven pancreatic enzymes of bovine (chymotrypsinogen A, trypsino- gen, carboxypeptidase A, RNase and DNase) or porcine (a-amylase and lipase) origins were obtained from Worthington Biochemicals, Freehold, New Jersey, USA and used as antigens. The antisera were

* Present address: Department of Anatomy, Universit6 de Montr6al, Montr6al, Canada

** Present address: First Department of Internal Medicine Niigata University, Niigata, Japan

raised in guinea pigs by subcutaneous injections of the emulsified anti- gens as described previously [7]. Each antiserum obtained was tested against the seven enzymes by the double-immunodiffusion technique on agar gel plates [7, 16]. The antisera were also tested against normal bovine pancreatic extract.

Preparation of the Tissues

Pancreatic tissues from seven healthy cattle were obtained at the Abat- toir Municipal, Geneva, just after slaughter. Samples from the splenic and duodenal parts of the pancreas were fixed by immersion in Bouin's solution. Small samples from the splenic part of the pancreas of nine diabetic cattle were obtained from the Istituto di Patologia Generale e Anatomia Patologica Veterinaria, Universit& degli Studie de Perugia, Italy. Diabetes had been induced in these animals by infection with the foot- and mouth virus [2, 3], and was characterized by emaciation, anorexia, polyuria, glycosuria, ketonuria and hyperglycaemia (12.98 _+ 0.38 versus 4.05 + 0.22 mmol/l). Clinical findings and labora- tory analysis which brought about the diagnosis of diabetes in these an- imals have been reported previously [2, 3]. The animals were slaugh- tered at different time points after the beginning of the infection and their pancreases were fixed by immersion in Bouin's solution.

Immunofluoreseence Technique

Normal and diabetic bovine pancreatic tissues fixed in Bouin's fluid for 24 h were embedded in paraffin. Sections (5 Ix) were deparaffinized, rehydrated and processed for the immunofluorescence technique as described previously [7]. The seven anti-exocrine enzyme antisera were applied on successive serial sections of the pancreatic tissue of each an- imal. The antisera were used at the following dilutions in 10 mmol phosphate buffered saline:anti-amylase 1:50, anti-chymotrypsinogen 1:100, anti-trypsinogen 1:100, anti-lipase 1:20, anti-carboxypepti- dase A 1:20, anti-RNase 1:20, anti-DNase 1:20.

0012 -186X/82 /0023 /0065 /$ 0 1 .0 0

66 M. Bendayan et al. : Pancreatic Enzymes in Diabetic Cattle by Immunohistochemistry

Fig. 1 a-b. Normal bovine pancreatic tissue incubated with a anti-chymotrypsinogen and b anti-amylase antisera. The fluorescent staining intensity is similar in most of the acinar cells in a, but in b the acini located around the islets of Langerhans (IL) are more brightly stained than the others. (X150). Parts e and d show serial sections of diabetic bovine pancreas incubated with c anti-trypsinogen and d anti-amylase antisera respectively. The acinar cells in e are brightly fluorescent while the same acini in d show only a weak staining. Surrounding the acinar cells is a non-reactive tissue which corresponds to loose connective tissue and tubular neoformations ( x 250)

The specificity of the immuno-staining was checked: (1) by adsorb- ing the antibodies with an excess of the corresponding antigen before applying to the sections; (2) by incubating the antisera with a saline ex- tract of normal bovine pancreas for 24 h at 4 ~ before applying to the sections; (3) by using non-immunized guinea-pig serum in the first step; and (4) by omitting the first incubation.

Results

The specificities of the anti-exocrine enzyme antibodies as shown previously with immunofluorescence [7] and immunoelectron microscopy [8, 17] were confirmed by

M. Bendayan et al.: Pancreatic Enzymes in Diabetic Cattle by Immunohistochemistry

Table 1. Fluorescence intensity in exocrine acinar cells of the diabetic bovine pancreas

67

Diabetic pancreas Enzymes

Animal Days from Amylase Lipase Carboxy- Chymo- Trypsinogen RNase DNase number the infection peptidase trypsinogen

1210 45 - weak weak + + + + 1211 65 weako r - w e a k o r - weak + + + + 1212 70 - weak + + + + + 1213 62 - weak + + + + + 1245 21 - weak weak + + - - 1246 16 weak or - - + + + + + 1247 12 weakor - w e a k o r - weako r - + + + + 1249 15 weak or - weak + + + + 1250 15 - weak weak + + weak +

Fig. 2. Diabetic bovine pancreatic tissue showing lympho- cyte infiltrations at the site of the endocrine tissue (ar- rows). The exocrine acinar tissue appears well preserved. (hematoxylin-eosin) ( x 150)

the double-immunodiffusion tests. A precipitin line was present at the site of reaction between the antibody and the specific antigen; a weak precipitin line was also present between the antibody and the pancreatic extract. No cross-reaction with the other antigens was observed.

By immunofluorescence, the acinar cells of the nor- mal bovine pancreas showed positive reactions. The staining was restricted to the cell cytoplasm (Fig. 1 a and b) and complete inhibition of the staining was obtained in the different control conditions. The endocrine cells of the islets of Langerhans were consistently negative (Fig. 1 b). In the tissue of some animals, the majority of acinar cells showed a positive reaction of similar intensi- ty but in most cases the staining pattern was not homo- geneous; the acini located close to the endocrine tissue were brightly fluorescent while others further from the islets showed a weak or even a negative reaction (Fig. 1 b). This inhomogeneity was found for all seven en- zymes tested, and in both the splenic and duodenal parts of the pancreas.

In the pancreatic tissue of the diabetic cattle, chymo- trypsinogen and trypsinogen were constantly present by immunofluorescence in the acinar cells while amylase, lipase and carboxypeptidase A gave a weak or negative

reaction. DNase and RNase were present in all but one diabetic pancreas. Figures I c and 1 d illustrate two serial sections of pancreatic tissue from a diabetic animal, ex- posed to anti-trypsinogen, and anti-amylase antisera re- spectively. The bright fluorescence obtained with the anti-trypsinogen (Fig. 1 c) was patchy due to unreactive regions corresponding to the oedematous and infiltrated interstitial tissue; the same acinar cells were only weakly stained by the anti-amylase antiserum (Fig. 1 d). A semi- quantitative evaluation summarizing these results is re- ported in Table 1. The pancreatic tissue of these different diabetic animals was characterized histologically by the disappearance of most of the islet structures and the presence of an inflammatory reaction with lymphocyte infiltration at the site of the endocrine tissue (Fig. 2). No evident correlation was found between these alterations, the disappearance of enzymes and the duration of the in- fection.

Discussion

When the enzyme content of normal and diabetic bovine pancreas was examined by the indirect immunfluores-

68 M. Bendayan et al.: Pancreatic Enzymes in Diabetic Cattle by Immunohistochemistry

cent technique, major differences were found with re- spect to three enzymes, amylase, lipase and carboxy- peptidase. The results from normal pancreas confirm previous work [12, 14, 17, 21], showing the presence of several secretory proteins in acinar cells and extend these data to include lipase and amylase. Since our amylase and lipase antigens were of porcine origin, the present re- sults indicate cross-reactivity between the porcine and bovine enzymes. In the normal bovine pancreas, as in rat pancreas [7], islets of Langerhans were surrounded by a halo of brightly fluorescent cells while those more distant from the islet were less intensely fluorescent. This may indicate the existence of a functional partition of the exocrine pancreas into peri- and tele-insular regions [7, 10, 13]. Furthermore, intercellular junctional specializa- tions were found between endocrine and exocrine pan- creatic cells, implying that both types of cells are struc- turally and functionally associated [5].

In contrast, the acinar cells of the diabetic bovine pancreas had markedly reduced contents of amylase, li- pase and carboxypeptidase, as reflected by a weak im- munofluorescent staining for these enzymes. These re- sults are consistent with our previous finding that, in the pancreas of streptozotocin-diabetic rats, the peri-insular exocrine tissue is devoid of amylase while chymotrypsi- nogen is normally distributed [6]. Biochemical studies have shown that the pancreatic amylase content de- creases in alloxan diabetic rats [4,18] and it is well known that insulin plays a key role in the synthesis of amylase [4, 18]. Recently, this effect has been shown to be due to a decreased cellular content of amylase messenger RNA in streptozotocin-diabetic rats - the normal mRNA con- tent being restored by insulin administration to these ani- mals [11]. From our results alone, however, it is not possi- ble to decide whether the observed alterations in pan- creatic enzyme content are due to a destruction of the en- docrine tissue or whether there is a direct viral effect on the exocrine cells.

Acknowledgements. We thank Dr. L. Orci for encouragement and sup- port and Drs. S. Weir and D. Brown for help in writing the manuscript. This work was supported by the Swiss National Science Foundation grant no. 3.668.80 and the Medical Research Council of Canada.

4. Ben Abdeljlil A, Palla JC, Desnuelle P (1965) Effect of insulin on pancreatic amylase and chymotrypsinogen. Biochem Biophys Res Commun 18:71-75

5. Bendayan M (1982) Contacts between endocrine and exocrine cells in the pancreas. Cell Tissue Res 222:227-230

6. Bendayan M, Ito S (1978) Immunocytochemistry of enzymes in normal and diabetic rat exocrine pancreas. Experientia 34:933 (Abstract)

7. Bendayan M, Ito S (1979) Immunhistochemical localization of ex- ocrine enzymes in normal pancreas. J Histochem Cytochem 27: 1029-1034

8. Bendayan M, Roth J, Perrelet A, Orci L (1980) Quantitative im- munocytochemical localization of pancreatic secretory proteins in subcellular compartments of the rat acinar cell. J Histochem Cyto- chem 28:149-160

9. Chey WY, Shay H, Shuman CR (1963) External pancreatic secre- tion in diabetes mellitus. Ann Intern Med 59:812-821

10. Hanson E (1959) The formation of pancreatic juice proteins studied with labelled amino-acids. Acta Physiol Scan 46: supp1161,1-99

11. Korc M, Overbach D, Quinto C, Rntter WJ (1981) Pancreatic islet- acinar cell interaction: amylase messenger RNA levels are deter- mined by insulin. Science 213:351-353

12. Kraehenbuhl JP, Racine L, Jamieson JD (1977) Immunocytochem- ical localization of secretory proteins in bovine pancreatic exocrine cells. J Cell Bio172:406423

13. Malaisse-Lagae F, Ravazzola M, Robberecht P, Vandermeers A, Malaisse W J, Orci L (1975) Exocrine pancreas: evidence for topo- graphic partition of secretory function. Science 190:759-797

14. Marshall JM Jr (1954) Distribution of chymotrypsinogen, procar- boxypeptidase, desoxyribonuclease and ribonuclease in bovine pancreas. Exp Cell Res 6:240-242

15. Orci L, Baetens D, Rufener C, Amherdt M, Ravazzola M, Studer P, Malaisse-Lagae F, Unger R (1976) Hypertrophy and hyperplasia of somatostatin containing D-cells in diabetes. Proc. Acad Sci USA: 1338-1342

16. Ouchterlony O (1958) Diffusion in gel, methods for immunological analysis. Prog Allergy 5 : 1-78

17. Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J His- tochem Cytochem 26:1074-1081

18. SOling HD, Unger KO (1972) The role of insulin in the regulation of amylase synthesis in the rat pancreas. Europ J Clin Invest 2: 199-212

19. Stefan Y, Malaisse-Lagae F, Yoon JW, Notkins AL, Orci L (1978) Virus induced diabetes in mice: a quantitative evaluation of islet cell population by immunofluorescence technique. Diabetologia 15: 395401

20. Vacca JB, Henke WJ, Knight WA (1964) The exocrine pancreas in diabetes mellitus. Ann Intern Med 61 : 142-147

21. Yasuda K, Coons AH (1966) Localization by immunofluorescence of amylase, trypsinogen and chymotrypsinogen in the acinar cells of the pig pancreas. J Histochem Cytochem 14:303-313

References

1. Baetens D, Stefan Y, Ravazzola M, Malaisse-Lagae F, Coleman DL, Orci L (1978) Alteration of islet cell populations in spontane- ously diabetic mice. Diabetes 27:1-7

2. Barboni E, Manocchio I (1962) Alterazioni pancreatiche in bovini con diabete mellito post-aftoso. Arch Vet Ita113:477490

3. Barboni E, Manocchio I, Asdruballi G (1966) Osservazioni sul dia- bete mellito del bovini da afta epizootica sperimentale. Vet Ita117: 339-368

Received: 13 October 1981 and in revised form: 17 February 1982

Dr. M. Bendayan Department of Anatomy Faculty of Medicine Universit6 de Montr6al C.P. 6128, Succ. A Montr6al, Qu6bec Canada, H3C 3J7